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Research Overview
Research in my lab focuses on five important areas at the interface between molecular cell biology and medicine:
A. Red blood cell development.
Red blood cell development, especially on regulation of proliferation and differentiation of several erythroid progenitor cells by extracellular signals, including erythropoietin and glucocorticoids, and by key microRNAs and transcription factors. We also study the mechanisms of chromatin condensation and enucleation;
Hematopoietic stem cells, identifying the stromal cells in the fetal liver and other organs that support stem cell self- renewal in vivo, and identifying novel growth factors that support stem cell expansion in culture;
MicroRNAs, defining their roles in differentiation of erythroid and lymphoid progenitor cells and in several hematopoietic cancers, and in regulating adipocyte and muscle differentiation and function;
D. Adiponectin and its paralogs.
Adiponectin, a hormone we cloned that is made exclusively by fat cells and that increases fatty acid and glucose metabolism by muscle, and several adiponectin orthologs that share adiponectin’s biological activities;
What ties all of these projects together is their focus on the basic cell and molecular biology of genes and proteins important for human physiology and disease.
Introduction Erythropoietin (Epo) is the principal regulator of red blood cell production; Epo is produced by the kidney in response to low oxygen pressure in the blood. Epo binds to Epo receptors on the surface of committed erythroid CFU-E progenitors, blocking apoptosis (programmed cell death), their usual fate, and triggering them to undergo a program of 4 – 5 terminal erythroid cell divisions and differentiation. We showed that the first two cell divisions, concomitant with differentiation from CFU-Es to late basophilic erythroblasts, are highly Epo-dependent; differentiation beyond this stage, involving chromatin condensation, ~1-2 terminal cell divisions, and enucleation, is no longer dependent on Epo but does require adhesion of the cells to a fibronectin matrix Following condensation of chromatin and subsequent enucleation reticulocytes (immature red cells) are released into the blood.
As evidenced by the properties of Epo- and EpoR- deficient mice we generated, Epo and the EpoR are essential for proliferation and differentiation of committed erythroid progenitors, as is the cytosolic protein-tyrosine kinase JAK-2. JAK2 binds to the EpoR cytosolic domain in the endoplasmic reticulum and facilitates its folding to promote cell surface expression. EpoRs exist as inactive dimers on the cell surface; Epo binding changes their conformation, leading to JAK2 transphosphorylation and activation.
JAK2 activates many signaling proteins including the transcription factor
Stat5, the PI-3’ kinase Akt kinase pathway, and the Ras MAPK pathway.
These pathways interact to prevent apoptosis of committed erythroid progenitors
allowing them to undergo a predetermined program of terminal proliferation
and erythroid differentiation. We showed that Stat5 directly activates
transcription of the anti-apoptotic protein bclxL. Stat5-/-
mice exhibit fetal anemia and increased apoptosis of erythroid progenitors
caused by reduced bclxL levels. Adult Stat5-/- mice
are anemic and deficient in generating high erythropoietic rates in response
to stress. Thus Stat5 controls one rate-determining step regulating early
erythroblast survival.
Activation of the PI-3’ kinase pathway leads to activation of the Akt kinase and then phosphorylation and inhibition of FOXO3a, a member of the Forkhead transcription factor family. FOXO3a, in turn, activates transcription of Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL). We showed that inhibition of TRAIL production by Epo addition partially rescues cells from apoptosis, demonstrating the importance of this pathway in red cell formation. Additionally, we showed that activated Akt phosphorylates the erythroid important transcription factor GATA-1 both in vitro and in vivo and enhances GATA-1 activity in erythroid cells.
The earlier committed progenitor, termed the burst- forming unit erythroid (BFU-E), can divide and generate additional BFU-Es (that is, undergo partial self- renewal) as well as generate later Epo- dependent CFU-E progenitors. Several cytokines and hormones are known to support BFU-E proliferation and formation of CFU-Es, including stem cell factor (SCF, the ligand for the c-kit protein tyrosine receptor) as well as IL-3, IL-6, and IGF-1. However, regulation of BFU-E proliferation and differentiation during basal and stress conditions is not well understood. We decided to focus on this important area based on the clinical observation that many bone marrow failure patients are helped by glucocorticoids (GCs) rather than Epo treatment. These patients already have very high Epo levels in the blood, but do not have sufficient Epo-responsive CFU-E cells in the bone marrow. As detailed below, we showed that GCs induce self-renewal of BFU-E cells, thereby protecting BFU-E cells from exhaustion, and in parallel increasing the number of CFU-E cells formed from each BFU-E.
Activation of Epo receptor signaling How Epo stimulation
activates EpoR-associated JAK2 is a central question in cytokine receptor
signaling. As a monomeric, asymmetric molecule, Epo employs two different
interfaces, termed site 1 and site 2, to bind to the two monomers of an
EpoR homodimer. Because the EpoR dimer is comprised of two identical transmembrane
proteins, it has been impossible to determine whether the EpoR bound to
Site 1 signals identically or differently from the one bound to Site 2.
Mala L. Radhakrishnan, a student in Professor Bruce Tidor’s lab
in the MIT Department of Bioengineering, computationally identified amino
acids in the EpoR important for binding to Site 1 in Epo but not site
2, and vice versa. She designed, and Lucy Zhang and Xiaohui Liu experimentally
created and verified, two mutant Epo receptors - one that binds only to
Epo site 1 but not site 2, and the other to site2 and not site 1. Expression
of either mutant receptor alone in Ba/F3 cells cannot elicit a signal
in response to erythropoietin, but when co-expressed in the same cell
there was a proliferative response and activation of the JAK2 Stat5 signaling
pathway. A truncated erythropoietin receptor with only one cytosolic tyrosine
(Y343; important for activation of Stat5) on only one receptor monomer
is sufficient for signaling in response to erythropoietin, regardless
of the monomer on which it is located.
Several years ago we identified three conserved hydrophobic residues in the juxtamembrane cytosolic domain of EpoR, L253, I257, and W258, and showed these are necessary for Epo- triggered activation of the associated JAK2. Mutating any of these residues to alanine in the EpoR homodimer dramatically suppressed growth and signaling responses to Epo stimulation but did not affect the ability of the Epo receptors to bind Jak2, traffic normally to the cell surface, or bind Epo. Using our Site-1 deficient and Site-2 deficient EpoRs we showed that these conserved residues could be located either on the Site 1- or the Site 2- binding receptor. We concluded that despite asymmetry in the ligand-receptor dimer interaction, both sides are competent for signaling, and we suggest that the receptors signal equally.
Mechanistic study of activation of normal and pathogenic Janus
kinase 2 and their association with the erythropoietin receptor
A point mutation in the Janus kinase 2 (JAK2) pseudo-kinase domain, V617F,
is found in most patients with Polycythemia Vera and half of those with
other myeloproliferative disorders. This mutation enables cytokine-independent
activation of JAK2 in cells that express a homodimeric cytokine receptor
such as the erythropoietin receptor (EpoR). The activation mechanisms
of both the normal and pathogenic JAK2 are poorly understood. Jiahai Shi
will shortly join the laboratory to study the interaction between JAK2
and the EpoR cytoplasmic domain by X-ray crystallography and in particular
determine the location of three conserved hydrophobic EpoR residues essential
for activation of normal JAK2 but interestingly not for activation of
JAK2-V617F. This work will be done in collaboration with Prof. Thomas
Schwartz of the MIT Biology Department. The long-term goal of this project
is to understand the activation mechanism of the normal and pathogenic
JAK2 as the foundation for drug design against JAK2-V617F-associated myeloproliferative
disorders.
Transcriptional control of gene expression during terminal erythroid differentiation. Shilpa Hattangadi’s project involves determining the transcriptional regulatory networks governing the important changes in gene expression that occur during terminal proliferation and differentiation of erythroid precursors. She began by using chromatin immunoprecipitation with antibodies specific for various erythroid- important transcription factors (ChIP), followed by hybridization of the recovered DNA to a promoter DNA microarray (ChIP-chip). She has since, in collaboration with Bill Wong and members of Rick Young’s laboratory, moved onto sequencing of the resulting DNA fragments (ChIP-Seq). This protocol enables her to determine all of the genes that have critical erythroid-important transcription factors bound to their promoter/ enhancer segments. Initial studies focus on transcriptional activation by Stat5, GATA1, FOG, and Foxo3, but other factors will soon be investigated. Shilpa’s long-term goal is to understand how the complex pattern of gene expression during terminal erythroid differentiation is regulated by transcription factors activated initially by signal transduction pathways downstream of the EpoR, but active in precursors no longer dependent on erythropoietin.
The second tier of this transcriptional network was evaluated by comprehensive expression profiling during erythroid differentiation: by isolation of mRNA from purified erythroid precursors in successive differentiation stages followed by hybridization to DNA microarrays and eventual confirmation of expression of selected genes by qRT-PCR. The results indicate that major changes in gene regulation occur during early erythroblast differentiation, concomitant with induction of Ter119 expression, an erythroid- specific surface protein, and hemoglobin. Upregulated genes include many expected categories such as those involved in hemoglobin metabolism, heme and porphyrin ring metabolism, cell and nuclear membrane structure, iron homeostasis, negative regulators of cell cycle, oxygen transport, and metabolism of oxygen and reactive oxygen species, among others. Genes that were significantly downregulated included genes involved in TNF-alpha production, NADP metabolism, NF-kappaB binding, actin binding, ubiquitin protein ligation, and non-erythroid specific functions such as immune responses and phagocytosis.
Along with her technical assistant, Karly Burke, Shilpa studied the effects of a specific kinase, Hipk2, which modulates the function of other transcription factors and cofactors. Hipk2 is highly induced during primary mouse fetal liver erythropoiesis and specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation – probably explained by cell cycle arrest as well as increased apoptosis – and terminal enucleation as well as the reduced accumulation of hemoglobin. Hipk2 knockdown reduces the expression of some genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis but does not affect the induction of several erythroid-specific transcription factors. This suggests that Hipk2 plays a significant role in terminal fetal liver erythroid differentiation and may regulate hemoglobin expression through noncanonical regulatory pathways.
Transcriptional changes, controlled by both covalent histone modifications and a strictly regulated network of gene expression, play a crucial role in red cell development. Bill Wong together with Shilpa, Lingbo Zhang and Manuel Ortega, an undergraduate student from the University of Puerto Rico, have performed mRNA-sequencing and chromatin-immunoprecipitation (Chip)-sequencing on primary murine fetal liver cells at various stages of erythroid differentiation.
mRNA-seq analysis accurately quantifies the absolute abundance of individual genes and also the fold changes at different developmental changes. Some of the abundantly expressed genes, in particular transcription factors such as GATA1, Sp2, FOG1 and LMO2, show less than 2 fold induction during erythroid differentiation, yet they are critical for erythropoiesis. Another class of highly expressed genes shows more than 10 fold induction during erythropoiesis, including hbb-b1, hbb-b2, Alas2, Band3, Darc, and Tmod1. Examination of abundant induced genes, which were not previously implicated in erythroid development, identified a number of novel stress hormone related receptors, transcription factors and serine/threonine kinases. Their functions are being studied using RNAi and chemical inhibitors.
Chromatin modifications, such as histone modifications, are critical to maintain a stable pattern of either gene activation or repression in cell fate specification and terminal differentiation. Bill performed Chip-seq on Ter119+ mouse fetal liver cells focusing on histone modifiers such as H3K4 trimethylation, H3K9 acetylation, H4K20 methylation, H3K27 trimethylation and also RNA polymerase II. H3K4 trimethylation, H3K9 acetylation, along with RNA polymerase II binding are generally associated with actively expressed transcripts such as Band3 and LMO2. However, some of the highly transcribed genes such as ferritin and Jag1 are only marked with H3K4 methylated, but not acetylated. Another interesting class of ‘trivalent’ genes are marked by H3K4 and H3K27 trimethylation and also H3K9 acetylation. He is currently investigating the effect of intensity and distribution of histone marks on the expression level of the target genes and their dynamic changes during erythroid differentiation and proliferation.
Mechanisms of stress erythropoiesis In situations of severe loss of red blood cells mammals and birds respond by a process known as stress erythropoiesis (SE). Johan Flygare hypothesizes that if the molecular pathways that induce SE are understood it will be possible to develop erythropoiesis stimulating agents that will complement or replace Epo treatment in anemic patients. Glucocorticoids (GCs) are known to be very potent enhancers of SE. This stimulatory effect of GCs on SE is utilized in the therapeutic regimen of Diamond-Blackfan Anemia (DBA), an erythropoietin-resistant congenital red cell aplasia. While an Epo-dependent balance of late red cell precursor survival normally maintains red cell homeostasis, Johan’s findings indicate that the physiology of SE involves a stimulation of earlier erythroid progenitors, which when activated are able to rescue red cell production in conditions such as DBA, where erythropoietin has little effect.
Johan showed that glucocorticoids stimulate self-renewal of early Epo-independent progenitor cells (burst-forming units erythroid or BFU-Es), increase production of colony-forming units erythroid (CFU-E) erythroid progenitors from the BFU-E cells, and enhance terminal erythroid differentiation. He first established two FACS-based methods to separate and purify BFU-E and CFU-E cells from mouse fetal liver. He demonstrated that GCs induce self-renewal of BFU-E cells, and not of CFU-E cells or erythroblasts. GCs thereby protect BFU-E cells from exhaustion, and in parallel increase the number of CFU-E cells formed from each BFU-E >10-fold. He further demonstrated that GCs do not inhibit, but rather stimulate erythropoietin-dependent terminal differentiation of freshly isolated erythroid CFU-E progenitors. He proposes a physiological model of stress erythropoiesis where increased levels of GCs help maintain the earliest erythroid progenitors, increase CFU-E output, and at the same time stimulate terminal differentiation, thus promoting both a rapid and long-lasting increase in red blood cell production.
Since the main action of the activated GCR is to interact with chromatin and regulate transcription Johan hopes to answer many questions by mapping exactly where in the chromatin the activated GCR binds by ChIP-Seq, which binding partners it has and how transcription is repressed and/or activated at these sites in BFU-E cells.
Chromatin condensation and enucleation in late stage erythroblasts Mammalian erythroid cells undergo enucleation during a late stage of differentiation, a process that does not occur in other vertebrates. This process has critical physiological and evolutional significance for the morphogenesis and hemoglobin enrichment of mature mammalian red blood cells. Although enucleation has been known for decades, the mechanisms that regulate this process remain obscure. Peng Ji is investigating the mechanism of mammalian erythroid cell enucleation. This and many of our other studies on EpoR signal transduction make use of the system Jing Zhang developed several years ago; purified fetal liver erythroid progenitors (mainly CFU-Es) are plated on fibronectin- coated dishes and cultured in the presence of Epo; they undergo normal terminal proliferation and differentiation that can be followed on a single cell level by FACS.
Since actin filaments have been shown to be critical for enucleation, Peng determined the role of different Rho GTPases, the master regulators of actin nucleation, on enucleation. Peng showed that deregulation of Rac GTPase during the late stages of erythropoiesis completely blocked enucleation of cultured mouse fetal erythroblasts without affecting normal proliferation and differentiation. The contractile actin ring formed on the plasma membrane of late-stage erythroblasts at the boundary between the cytoplasm and nucleus of enucleating cells was disrupted when Rac GTPase was inhibited in late stages of erythropoiesis. Peng further demonstrated that Rac GTPase activity is mediated by the downstream target protein, mDia2, a formin required for nucleation of unbranched actin filaments. These results reveal important roles for Rac GTPase and mDia2 in enucleation of mammalian erythroblasts.
In collaboration with Tzutzuy Ramirez, a fellow with Dr. Maki Murata Hori of the Temasek Life Sciences Laboratory, Singapore, and Senthil Jayapal, Peng is investigating the roles of many cytoskeletal and other proteins in nuclear migration and enucleation of these cells, in part using video microscopy of cells expressing fluorescent- tagged proteins. Initial results show that, unlike conventional cytokinesis, the nucleus is squeezed out by formation of a bleb-like protrusion from a limited area of the erythroblast cell cortex; the bleb increases in size by dynamic contractions of asymmetrically distributed actomyosin. Concomitant with displacement of the nucleus to one side of the cell, actin and myosin II became asymmetrically enriched in the incipient reticulocyte of the erythroblast where dynamic and random contractions occur.
Peng, assisted by Victor Yeh, is also focusing on the role of histone deacetylases (HDACs) in chromatin and nuclear condensation and enucleation of late erythroid cells. They showed that inhibition of HDAC activities by Trichostatin A completely blocks enucleation, and that knockdown of certain specific HDAC mRNAs partially blocks enucleation. Peng further showed that mDia2 is acetylated in vivo and his current aim is to determine whether HDAC6 can deacetylate mDia2 and in so doing activate this formin and thus promote red cell enucleation. In parallel they are investigating the roles of HDACs in inactivating gene transcription and condensing chromatin prior to enucleation.
At the same time, Peng is also interested in the roles of mDia1 and mDia2 on hematopoietic stem cell homing and migration. He is currently generating an mDia2 knockout mouse model and he will use these mice to study the roles of mDia2 in hematopoiesis.
Myc, Chromatin condensation, histone acetylation, and enucleation in late stage erythroblasts Using the in-vitro erythroid culture system developed in the Lodish lab, Senthil Raja Jayapal, who is a joint graduate student in the Lodish lab and in the labs of Bing Lim at the Genome Institute of Singapore and Philipp Kaldis at Institute of Molecular and Cell Biology in Singapore, is investigating the role of cell cycle proteins and epigenetic modifications of histones in late stage erythroid maturation. In order to study the relationship between proliferation and differentiation programs during terminal erythroid maturation, he initially chose to focus on c-myc, which directs proliferation in many cell types and is down regulated during terminal differentiation when cells withdraw from cell cycle. The protein levels of c-myc are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G1-phase and enucleation, suggesting possible roles for c-myc in one or both of these processes. Surprisingly, ectopic c-myc expression had a dose dependent effect on terminal erythroid maturation. Ectopic expression of c-myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown, but specifically blocked erythroid nuclear condensation and enucleation. When over-expressed at levels much higher than physiological, c-myc blocked erythroid differentiation completely and the cells continued to proliferate in culture with an early erythroblast morphology. These studies revealed important roles for c-myc in erythroid cells independent of its cell cycle regulatory functions.
Since histone deacetylation has been associated with erythroid nuclear condensation and enucleation, he compared the changes in acetylation status of histones H3 and H4 in erythroid cells with physiological levels of ectopic myc expression that are specifically blocked in enucleation, relative to the untreated wild type erythroblasts. c-myc was able to prevent deacetylation at several lysine residues that are normally deacetylated during erythroid maturation. By transcriptional profiling, one specific histone acetyl transferase (HAT) was shown to be upregulated by ectopic myc expression. The level of this HAT, like that of c-myc, normally decreases dramatically during late stage erythroid maturation. Chromatin immunoprecipitation assays demonstrated binding of c-myc to the promoter region of this HAT, indicating that it is a direct myc target in erythroid cells. Over-expression of this HAT inhibits nuclear condensation and enucleation specifically without affecting other aspects of terminal erythroid differentiation, and prevents histone deacetylation similar to ectopic c-myc expression. These data support a model where histone deacetylation associated with down regulation of c-myc and this HAT is essential for chromatin condensation and enucleation in mammalian erythroid cells. Currently, he is investigating the roles of other HATs and HDACs in terminal erythroid differentiation.
Diamond Blackfan anemia Diamond Blackfan Disease (DBA) is a congenital anemia that develops at birth or soon after. Anemia is due to failure of production of erythrocytes and their precursors, with normal or near normal myeloid cells and platelets. DBA is inherited in about 10-20% of cases, mostly as an autosomal dominant. Genetic studies have led to the surprising identification of mutations in a ribosomal protein (RP) gene, RPS19, in 20 - 25% of both familial and sporadic cases. Recently Colin Sieff and colleagues discovered a haploinsufficient mutation in another ribosomal protein gene, RPS24, which co-segregates with affected family members in a large DBA pedigree, and has also identified mutations in three large subunit RPs. While experiments in yeast and mammalian cells show that RPS19 depletion or mutation leads to a block in ribosomal RNA biosynthesis, this result does not explain why erythropoiesis is so severely affected in DBA.
One hypothesis is that during fetal development immature erythroid cells
proliferate more rapidly than other lineages and therefore require very
high ribosome synthetic rates to generate sufficient capacity for translation
of erythroid specific transcripts that must take place before these unique
cells enucleate. To test this hypothesis Colin, assisted by Jing Yang,
first studied proliferation and differentiation of mouse CFU-Es in culture.
During the first 24 hours of culture in the presence of Epo the cell number
increases 3-4 fold. Remarkably, there is a 6-fold increase in RNA content
during the same period, suggesting that the cells accumulate an excess
of ribosomal RNA and ribosomes during early erythropoiesis. siRNA RPS19
knockdown cells showed reduced proliferation at 48 hours of culture, but
the differentiation pattern of the surviving knockdown positive cells
was similar to that of the controls.
While RPS19 mRNA is rapidly depleted by an siRNA, Western analysis did not show a deficiency of RPS19 protein. This suggests strongly that the proliferative defect is not due to insufficiency of RPS19 protein, and is more likely due to a block in ribosome biogenesis that leads to nucleolar stress. When earlier erythroid progenitors (BFU-Es) were infected by a retrovirus encoding an RPS19siRNA, the cells showed reduced proliferation but normal differentiation, and cell cycle analysis showed a G1/S phase delay. Colin then determined that p53 protein is increased in the knockdown cells, and the mRNA level for p21, a transcriptional target of p53, is also increased. Furthermore, he showed that the MYB protein is decreased after knockdown of RPS19, and that KIT mRNA, a transcriptional target of MYB, is also reduced, as is the amount of cell surface KIT protein. Thus RPS19 insufficient erythroid cells may proliferate poorly because of p53 mediated cell cycle arrest, and also because of decreased expression of the key erythroid signaling receptor KIT.
Introduction Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and to differentiate into all blood cell types – erythroid, myeloid, and lymphoid cells. These very rare cells – about 1:10,000 in fetal liver and bone marrow - form the basis of bone marrow transplantation for treatment of leukemia and other cancers, and are also a promising cell target for developing gene therapies for treating a broad variety of human diseases. However, development of these important clinical applications of HSCs are greatly hampered by the lack of understanding of the extracellular and intracellular signals that govern their fates and the difficulty in ex vivo expansion of these cells. We quantitate these cells by bone marrow transplantation, monitoring long- term repopulation of the hematopoietic compartment of lethally irradiated mice. This assay requires several months to complete.
Novel growth factors for hematopoietic stem cells No
single known growth factor or combination of growth factors reproducibly
supported HSC expansion in culture. Furthermore existing lines of “supportive
stromal cells” did not support expansion of HSCs; at best they maintained
the level of HSCs over time, presumably due to a steady state between
generation of new HSCs by division and differentiation of “old”
stem cells. Thus, several years ago Chengcheng (Alec) Zhang turned to
mouse fetal liver since the number of fetal HSCs normally increased markedly
between embryonic Day 14 and Day 21. Chengcheng hypothesized that unknown
growth proteins are produced by as- yet unidentified populations of fetal
liver cells that stimulate the expansion of fetal liver HSCs. He identified
Embryonic Day 15 fetal liver CD3+ Ter119- cells as a novel cell population
that supports a net expansion of HSC numbers in culture. By transcriptional
profiling of these cells and several others that do not support HSC expansion,
Chengcheng uncovered several novel growth factors that, together, supported
an unprecedented extent of ex vivo expansion of bone marrow HSCs: insulin
- like growth factor 2 (IGF - 2) and Angiopoietin-like 2 and 3. A serum-
free medium containing only low levels of stem cell factor (SCF), thrombopoietin
(TPO), IGF-2, FGF-1, and Angiopoietin-like 2 or 3 stimulated a 24-30-fold
expansion of HSCs following 10 days of culture of highly enriched mouse
stem cells. A similar “cocktail” of five growth factors in
serum- free medium supported a ~30- fold expansion of human hematopoietic
cord blood stem cells, and we are currently collaborating William Hwang
at the Singapore General Hospital to carry out preclinical and eventually
clinical studies on ex vivo cord blood HSC expansion.
Supportive stromal cells for hematopoietic stem cells Hematopoietic stem cell environments or niches are very important in determination of HSC self-renewal and differentiation; fibroblasts, endothelial cells, and osteoblasts have been postulated as important constituents and regulators of HSC niches in the bone marrow. Song Chou, together with Yachao Liu, are trying to characterize the stromal cells that support HSC expansion in fetal liver. By using real-time PCR, Song discovered that fetal liver CD3+ cells not only are highly enriched for Angptl3 and IGF2 mRNAs, but also for the mRNAs encoding stem cell factor (SCF), the membrane- anchored ligand for the c-kit tyrosine kinase receptor, and TPO mRNAs. Since these four hormones form a complete set of growth factors that are able to significantly expand HSCs ex vivo, it is likely that fetal liver CD3+ cells are able to secrete all four crucial growth factors and are able to support HSC expansion in fetal liver in the absence of other added cytokines.
Because of the low apparent level of CD3 expressed on these fetal liver
cells and because we were unable to detect expression of the protein targeted
by the monoclonal anti- CD3 antibodies, we sought other methods to purify
these presumed stromal cells. SCF is fabricated as a transmembrane plasma
membrane protein that normally binds to its receptor, c-Kit, on the surface
of adjacent cells; since all HSCs in fetal liver express c-Kit, these
stromal cells may be located in close proximity to HSCs and interact with
HSCs through SCF. Song further discovered that these potential stromal
cells also highly express DLK, another membrane-bound cytokine that is
involved in the maintenance and self-renewal of HSCs. Using flow cytometry,
he showed about 1-2% of total fetal liver cells are SCF+DLK+ and that
the vast majority of SCF+ cells are also DLK+. He purified SCF+DLK+ cells
by flow cytometry and found the mRNAs of seven relevant HSC expansion
factors are enriched more than 50 fold over SCF-DLK- cells, which represent
the majority of the fetal liver cells. Thus by sorting for SCF+DLK+ surface
phenotype, he was able to highly enrich the stromal cells for HSC expansion
in fetal liver. Song also used immnunocytochemistry to show that the majority
of SCF+ cells also express DLK and Angptl3. Thus these cells are highly
homogeneous and likely are the principle stromal cell population for HSCs
in fetal liver. He is currently further characterizing these cells and
determine other signaling molecule they produce that affect HSC expansion.
Introduction MicroRNAs (miRNAs) are small endogenous ~22-nt non-coding RNAs that base pair to sites within target mRNAs, triggering either a block in translation or mRNA degradation or both. The expression of miRNAs is often tissue-specific or developmental-specific. As shown by the Bartel laboratory and others, humans have over 600 genes that encode miRNAs, an abundance corresponding to almost three percent of protein-coding genes; computational and experimental analyses suggest that miRNAs may regulate expression of ~30% of human and mouse genes. Based on the evolutionary conservation of many miRNAs among different animal lineages, it is reasonable to suspect that some mammalian miRNAs have important conserved functions in cellular development function. Indeed, the post-transcriptional programs controlled by specific miRNAs affect diverse biological processes, including development, cell differentiation, apoptosis, immune responses, metabolism and many diseases including various cancers, cardiovascular disease, viral infection and neurodegenerative diseases.
MicroRNAs in fat cell development and obesity Huangming
Xie is examining the role of miRNAs in adipogenesis using several adipocyte
cell culture differentiation systems. He profiled miRNA expression during
in vitro adipogenesis of the preadipocyte 3T3-L1 cells using miRNA microarrays
and validated by RT-PCR eight miRNAs that are significantly upregulated
and four that are downregulated. Similar changes in miRNA expression were
observed by comparison of mature primary adipocytes and enriched primary
preadipocytes. He also profiled miRNA expression in purified mature adipocytes
and compared miRNA profiles in epididymal adipocytes from normal and leptin
deficient or diet-induced obese mice. Importantly, miRNAs that were induced
during adipogenesis were downregulated in adipocytes from both types of
obese mice and vice versa. These changes are likely associated with the
chronic inflammatory environment in obese adipose tissue as they were
mimicked by TNF-alpha treatment of differentiated adipocytes.
Huangming, Lei Sun and Lingbo Zhang, are investigating the role of these candidate miRNAs in adipogenesis and in functions of mature adipocytes. Ectopic expression of two adipocyte-enriched miRNAs in preadipocytes accelerated adipogenesis, as measured both by the upregulation of many adipocyte-important genes including adiponectin and the key transcription factor PPAR-gamma, and by an increase in triglyceride accumulation at an early stage of adipogenesis.
Together, these studies present a comprehensive view of miRNA dynamics in adipocyte development and function and provide an important first step towards construction of the entire RNA regulatory network underlying fat cell development and adipose dysfunction in obesity. These findings have important implications for the understanding the link between chronic inflammation and obesity with insulin resistance. An understanding of the role of miRNAs in adipose biology, obesity and related medical complications such as insulin resistance may lead to novel RNA-based therapies that complement current anti-obesity treatments.
Huangming and Lei have also profiled the genome-wide miRNA expression patterns of white fat, brown fat, and skeletal muscle. Interestingly, many “myogenic” miRNAs were abundant in brown fat but absent in white fat, which is consistent with the current notion that during lineage specification the brown adipocyte precursor is closer to the muscle precursor than the white adipocyte precursor. They identified several miRNAs that were preferentially expressed in only one of the three tissue types. Strikingly, ectopic expression of one brown fat- enriched miRNA in myogenic C2C12 cells inhibited myotube maturation, as evidenced by absence of key muscle morphological changes and also reduced expression of key myogenic proteins. In addition, ectopic expression of the same miRNA in 3T3-F442A white preadipocytes induced several key mRNAs unique to brown fat cells. Taken together, these results underlie the importance of tissue enriched miRNAs in regulating lineage specification between brown fat and muscle, and also suggest that certain miRNAs may have therapeutic potential in inducing expression of brown fat-specific genes.
A microRNA important for neurogenesis that targets p53 Minh Le,
a graduate student working jointly with our lab and that of Bing Lim in
the Genome Institute of Singapore, has elucidated the role of miR-125b
in neurogenesis. miR-125 is a homolog of lin-4, which is important for
developmental timing in C. elegans. The expression of miR-125b is upregulated
during embryogenesis and enriched in the nervous system of vertebrate
species. However, the functions and targets of miR-125b in these species
remain poorly understood. Minh first obtained the expression profile of
microRNAs during neuronal differentiation of the human neuroblastoma cell
line SH-SY5Y; six microRNAs were significantly upregulated during differentiation
induced by all-trans-retinoic acid and brain-derived neurotrophic factor.
She then demonstrated that ectopic expression of either miR-124a or miR-125b
increases the percentage of differentiated SH-SY5Y cells with neurite
outgrowth. Subsequently, she focused her functional analysis on miR-125b
and demonstrated the important role of this miRNA in both spontaneous
and induced differentiation of SH-SH5Y cells. miR-125b is also upregulated
during differentiation of human neural progenitor ReNcell VM cells, and
Minh showed that miR-125b ectopic expression significantly promotes neurite
outgrowth of these cells. To identify the targets of miR-125b regulation,
Minh profiled the global changes in gene expression following miR-125b
ectopic expression in SH-SY5Y cells. More than 50% of the downregulated
mRNAs contain the seed match sequence of miR-125b; transcripts with stronger
seed matches were repressed to a greater extent. Importantly, TargetScan
5.1 predicted 188 of the downregulated transcripts to be direct targets
of miR-125b. Pathway analysis suggests that a subset of miR-125b-repressed
targets antagonize neuronal genes in several neurogenic pathways, thereby
mediating the positive effect of miR-125b on neuronal differentiation.
Minh further confirmed the binding of miR-125b to the microRNA response
elements of nine selected mRNA targets and validated the binding specificity
for three targets. Together, these data reveal for the first time the
important role of miR-125b in human neuronal differentiation.
Furthermore, Minh demonstrated that miR-125b is indispensable for zebrafish
embryogenesis, particularly for the survival of neural cells during development.
She identified p53, a key tumor suppressor, as a bona fide target of miR-125b
in both zebrafish and humans. miR-125b-mediated downregulation of p53
is strictly dependent on the binding of miR-125b to a microRNA-response
element in the 3’ UTR of p53 mRNA. Overexpression of miR-125b represses
the endogenous level of p53 protein and suppresses apoptosis in human
neuroblastoma cells and human lung fibroblast cells. By contrast, knockdown
of miR-125b elevates the level of p53 protein and induces apoptosis in
human lung fibroblasts and in the zebrafish brain. In zebrafish this phenotype
can be rescued significantly by either ablation of endogenous p53 function
or by ectopic expression of miR-125b. Interestingly, miR-125b is downregulated
when zebrafish embryos are treated with gamma-irradiation or camptothecin,
corresponding to the rapid increase in p53 protein in response to DNA
damage. Ectopic expression of miR-125b suppresses both the increase of
p53 and stress-induced apoptosis. Minh also identified seven additional
targets of miR-125b in the p53 network and mapped the connections of miR-125b
to many other components of this network. Together, her study provides
a global view of miR-125b function, as an integrated component of the
cellular regulatory network, in modulating gene expression to maintain
the homeostasis of cell survival, death and differentiation during development.
MicroRNAs that modulate hematopoiesis As a first step towards testing the idea that miRNAs might play roles in mammalian development, and more specifically hematopoiesis, Chang- Zheng Chen in collaboration with David Bartel, cloned about 100 unique miRNAs from mouse bone marrow. Three, miR-181, miR-223, and miR-142s, were exclusively or preferentially expressed in hematopoietic tissues. miR-181 was very strongly expressed in thymus, the primary lymphoid organ, which mainly contains T-lymphocytes. Mature miR-181 expression in the bone marrow cells was up-regulated in differentiated B-lymphocytes. Using retrovirus vectors he developed, Chang- Zheng ectopically expressed miR-181 in a population of bone marrow hematopoietic stem and progenitor cells. This led to an increased fraction of B-lineage cells both in tissue-culture differentiation assays and in transplanted adult mice; there was a corresponding decrease in CD-8+ T cells. These and other results with other miRNAs indicate that microRNAs are components of the molecular circuitry controlling mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. Some of this work is being done in Chang- Zheng’s own laboratory at the Stanford University School of Medicine.
MicroRNAs that modulate erythropoiesis Lingbo Zhang’s project involves identification of functionally important microRNAs during erythropoiesis by a retrovirus mediated microRNA overexpression functional screen. In collaboration with Bill Wong, and taking advantage of our in vitro erythrocyte progenitor culture and differentiation system, Lingbo used retrovirus infection to overexpress many erythroid lineage - enriched microRNAs in mouse E14.5 fetal liver Ter119 negative cells, followed by FACS analysis after two days of culture. Several microRNAs have marked effects on different aspects of erythropoiesis, including induction of erythroid genes, enucleation, survival, and proliferation. Lingbo is planning to knock down these microRNAs by virus - mediated microRNA sponge infection. Further analysis will be focus on how these microRNAs regulate erythropoiesis, such as computational identification as well as experimental validation of microRNA targets and upstream regulators. These discoveries will shed light on post-transcriptional regulation of erythropoiesis.
MicroRNAs that modulate myeloid development and leukemias Myelodysplastic
syndromes (MDS) and acute myeloid leukemia (AML) are frequently associated
with chromosomal translocations. Most of them involve oncogenes or transcription
factors that are up regulated or that form part of chimeric genes. The
t(2;11)(p21;q23) translocation is observed in cases of MDS and AML and
in her previous laboratory in Toulouse Marina Bousquet showed that this
translocation triggers upregulation of miR-125b. This was the first description
of microRNA deregulation by a chromosomal translocation, and implied that
AML and MDS carrying the t(2;11) translocation represent a new clinico-pathological
entity. Cell culture experiments demonstrate that miR-125b per se is able
to block the myeloid differentiation of human cell lines under various
stimulations. Since lin-4, the miR-125b ortholog in Caenorhaditis elegans,
is implicated in several developmental process, she hypothesizes that
deregulation of miR-125b expression would impair human and mouse haematopoiesis.
To check if the overexpression of miR-125b blocks myeloid differentiation and/or causes leukemias in vivo, Marina is using a retroviral construct encoding miR-125b to infect enriched hematopoietic stem/ progenitor cell populations. These cells are then injected into lethally irradiated recipient mice. With this mouse model, we will be able to investigate the effect of miR-125b overexpression on the establishment of different hematopoietic lineages and on the formation of hematopoietic cancers.
To better understand the role of miR-125b in hematopoiesis Marina is trying to identify miR-125b targets. She is using two experimental in vitro models: NB4 (human promyelocytic cell line) and 32Dcl3 (mouse promyelocytic cell line). NB4 and 32Dcl3 can be induced to differentiate to granulocytic cells with retinoic acid and G-CSF respectively. She is analyzing the total cellular gene expression pattern by both RQ-PCR and mRNA-seq, comparing undifferentiated and differentiated myeloid cell lines expressing or not miR-125b. She is focusing on mRNAs downregulated both in differentiated and undifferentiated cells by miR-125b overexpression, and determining whether these mRNAs are significantly enriched in those containing a potential miR-125b target segment in their 3’ UTR. It is likely that some of these specifically downregulated miR-125b targets will be recognizable because of their importance in myeloid differentiation, and these will be pursued further. Diu Nguyen, a visiting graduate student, is working with Marina to identify miR-125b targets using reporter assays to validate putative miR-125b target segments in these mRNAs. The combination of these different approaches should allow us to identify new miR-125b targets.
As noted below, recent work from our lab indicates that mir-125b is a novel bona fide negative regulator of p53 in human and zebrafish. One hypothesis is that miR-125b downregulation of p53 in some specific hematopoietic cell facilitates development of leukemic cells. However, p53 is not a conserved target among all vertebrate species and in particular the binding site for miR-125b is not conserved in mouse p53 mRNA. We hypothesize that even if the miR-125b binding site is not conserved in mouse p53, the p53 pathway is regulated by miR-125b in both human and mouse.
MicroRNAs affecting drug- resistance of leukemias Acute lymphoblastic leukemia (ALL) is one of the most common malignancies of children and young adults. Ai Kotani has identified and characterized specific miRNAs that are critical in the development and progression of MLL related leukemias, an ALL that shows poor prognosis. In MLL related ALL, expression of many miRNAs is downregulated, raising the possibility that downregulation of some miRNAs plays a critical role in the pathogenesis of this disease. miR-128 is one of these. Ai Kotani and Daon Ha, a UROP student, studied RS4; 11 cells, a cell line derived from a MLL-AF4 ALL patient in which a balanced translocation between the MLL and AF4 genes has occurred. RS4; 11 cells have a novel A to G point mutation in the miR-128b gene segment; this mutation is transcribed in the primary miR- 128 transcript and blocks the processing of the miR-128 precursor to mature miRNA. Like MLL related ALL, the RS4; 11 cell line is resistant to glucocorticoid- induced apoptosis. Ai and Daon showed that overexpression of the wild- type miR-128 gene in these cells restored glucocorticoid- induced apoptosis. Target genes directly downregulated by miR-128b include MLL, AF4, and both MLL-AF4 and AF4-MLL fusion genes. miR-221 normally downregulates the CDKN1B gene, which encodes the p27 cell cycle inhibitory protein. Overexpression of miR-221 also restored glucocorticoid- induced apoptosis to RS4; 11 cells and functioned synergistically with miR-128. These results demonstrate that downregulation of miR-128b and miR-221 induces glucocorticoid resistance, and that restoration of their levels is a promising therapeutic in MLL-AF4 ALL.
MicroRNAs affecting B cell development and function Beiyan Zhou
has used microRNA microarrays developed in the Bartel laboratory and Northern
Blot analysis to identify several miRNAs specifically upregulated in isolated
populations of thymic and splenic hematopoietic cells: B cells, immature
CD-4- CD-8- and CD-4+ CD-8+ T cells, as well as in more mature thymic
CD-4- CD-8+ and CD-4+ CD-8- T cells. She has confirmed these results by
Northern blotting. Based on these results, five micro RNAs (miR-195, miR-150,
miR-106, miR-181a, and miR142) were chosen for further analysis. miR-150
and miR-181a were highly expressed in the thymus and spleen, the major
secondary lymph organ, leading to our hypothesis that these two miRNAs
are involved in lymphopoietic regulation.
To study the potential regulatory roles of these miRNAs in T cell function Beiyan and Stephanie Wang, a UROP student, used retroviral infection of two clonal CD8+ T-cell lines to establish 8 stable clonal CD8+ T-cell lines that ectopically express either miR-106, miR-142, miR-150, or miR-195. She is currently establishing CD4 clonal cell lines that overexpress these selected miRNAs. These infected T-cell clones will be used for experiments to examine the effects of miRNAs on mature T-cell function. Meanwhile, Beiyan also discovered a group of micro RNAs that have been up or down regulated during T cell activation. The regulatory functions of these micro RNAs are under investigation now.
Beiyan also studied the expression patterns of these selected microRNAs in detail during several various developmental stages of both B and T cells. miR-150 is mainly expressed in the lymph nodes and spleen, and is highly upregulated during the development of mature T and B cells. In particular, expression of miR-150 is sharply upregulated at the immature B cell stage. In contrast, expression of miR-181 is sharply downregulated during B cell development, and several years ago Chang- Zhen Chen showed that overexpression of miR-181 in hematopoietic stem/ progenitor cells led to a significant increase in production of mature B cells. To explore the roles of miR-195 and miR-150 in lymphopoiesis Beiyan generated retroviral constructs that express each of these miRNAs. Ectopically expressed miR-150 (but not miR 195) elicited a significant inhibition of B cell development, and had little effect on other hematopoietic lineages. Further analysis showed that miR 150 overexpression did not affect B cell lineage commitment, as evidenced by unaltered pro-B cell numbers both 4 and 16 weeks after transplantation. Overexpression did block the pro-B to pre-B transition in the bone marrow. We hypothesize that premature expression of miR-150 in hematopoietic stem/progenitor cells inhibits production of proteins specifically required for early B cell development.
Several potential miR-150 targets were predicted by Target Scan screening. The mechanism of miR-150 regulation in B cell development is being further investigated by studying these potential targets, in particular B lineage specific transcription factors. One of the important transcription factor targeted by miR-150 is Myb, which is also an important regulator for B cell development. Mice with the myb gene selectively deleted in the B cell lineage have a B cell development deficiency. In order to test the direct regulatory relation between miR-150 and Myb, Beiyan is generating a knock in mouse model with potential miR-150 target sites mutated in the Myb 3’UTR region. This will provide us with important information on the role of miR-150 regulation of myb gene expression during B cell development. Other potential targets of this micro RNA at this stage are also being investigated. Additionally, Andrew Shie, a UROP student, is working with Beiyan to further investigate the regulatory role of miR-150 in early B cell development. Integrating computational and luciferase reporter assays, Andrew has identified several genes that are also downregulated by miR-150 in early B cell development. Direct regulation of these targets by miR-150 is been tested using several lymphatic cell lines. Cell proliferation and apoptotic regulation by these newly identified regulators are investigated.
In addition to early B cell development, Beiyan profiled microRNA expression during terminal B cell development. Upon T-cell dependent and T-cell independent stimulation, Beiyan found that a group of micro RNAs, including miR-150, are actively regulated; the roles of these micro RNAs in B cell activation will be further studied using both in vivo and in vitro systems. This work will continue in Beiyan’s own laboratory at Texas A&M University.
MicroRNAs affecting muscle differentiation and function Prakash Rao started his studies in the laboratory by identifying three microRNAs, miR-1, miR-133 and miR-206, that are upregulated during the differentiation of the C2C12 myoblast cell line into myotubes. In a collaborative study, Prakash (along with Dr. Roshan Kumar of the Young lab at Whitehead) identified the myogenic transcription factors responsible for their specific regulation during myogenesis.
The observation that miR-1, miR-133 and miR-206 are induced during muscle differentiation of a myoblastic line have led them to consider their potential mRNA targets in muscle differentiation. In particular, they have focused on the ability of miR’s to target components of conserved regulatory cascades that inhibit myogenesis, and on the regions within the 3` UTRs of these mRNAs that possesses miR-1 binding sites. Prakash and Greg Hyde, a visiting scientist, showed that miR-1 can inhibit reporter genes bearing regions of some of these potential target mRNAs. Overall, these studies point to the importance of miR-1 in promoting the myogenic program. Consistent with these hypotheses, Prakash, with able assistance from a UROP, Lauren Shields, has demonstrated the ability of miR-1 and miR-206 to inhibit proliferation and promote differentiation of a muscle-derived rhabdomyosarcoma cell line. Taken together with the observation that miR-1 and miR-206 levels are lower in muscle-derived tumors (data from Janet Shipley at the Institute for Cancer Research in the UK), it is likely that miR-1 and miR-206 are tumor suppressors in rhabdomyosarcoma cells.
Prakash, in collaboration with Drs Robert Blelloch (UCSF) and Rudolf
Jaenisch, also studied the impact of the global loss of all microRNAs
by deleting, specifically in muscle tissue, dgcr8, a gene required for
microRNA biogenesis. Lack of dgcr8 leads to a reduction in mature microRNAs
in the heart and skeletal muscle and leads to the development of heart
failure. This fully penetrant phenotype began with left ventricular malfunction
and progressed to a dilated cardiomyopathy and premature lethality. Taken
together, these observations reveal a critical role for microRNAs in maintaining
cardiac function in mature cardiomyocytes and raise the possibility that
only a handful of microRNAs maybe ultimately be responsible for the dramatic
cardiac phenotype seen in the absence of dgcr8.
Introduction In 1995 we cloned adiponectin, originally called Acrp30, as a novel adipocyte- specific secreted protein hormone. Adiponectin addition potently elevates fat and glucose catabolism by muscle, enhances glycogen accumulation in muscle, and inhibits gluconeogenesis in liver. Mutations in the adiponectin gene are linked to development of adult- onset diabetes and the levels of adiponectin in serum are reduced in obese and diabetic patients and mice. Circulating adiponectin levels negatively correlate with human plasma triglyceride and fasting insulin levels and several clinical studies showed that persons with low adiponectin levels are more likely to develop type II diabetes mellitus and cardiovascular disease. This data suggests that adiponectin is a potential determinant of insulin sensitivity.
Adiponectin has four domains: a cleaved amino-terminal signal sequence, a region without homology to known proteins, a collagen-like region, and a globular segment at the carboxy-terminus. The three-dimensional structure of the globular domain is strikingly similar to that of TNF -alpha even though there is no homology at the primary sequence level. Like TNF-alpha the globular domain forms homotrimers, and intermolecular disulfide bonds generate hexameric and high molecular weight Adiponectin species.
In collaboration with the Ruderman laboratory at B. U. Medical School,
we showed several years ago that treatment of rat striated muscle with
trimeric adiponectin led to phosphorylation and activation of AMP-activated
protein kinase (AMPK), an enzyme that when activated causes increases
in muscle fatty acid oxidation, glucose uptake and oxidation, and insulin
sensitivity. Adiponectin- mediated activation of AMPK caused phosphorylation
and thus diminished activity of acetyl CoA carboxylase, a corresponding
decrease in the concentration of malonyl CoA, and a corresponding increase
in long- chain fatty acid oxidation. In addition, adiponectin caused an
increase in glucose uptake by muscle. Both in vivo and in muscle culture
adiponectin most likely exerts its actions on muscle fatty acid oxidation
by inactivating ACC, via activation of AMPK and perhaps other signal transduction
proteins.
Activation of AMP kinase by adiponectin and insulin Adiponectin has important roles in enhancement of insulin sensitivity and these beneficial effects are closely associated with the activation of AMP-activated protein kinase (AMPK) in muscle and liver. How adiponectin activates AMPK is not known. Interestingly, AMPK activation by adiponectin is accompanied by an increase in concentration of 5’AMP, which implies the presence of signal transduction proteins in a pathway connecting the adiponectin signal with AMPK. Qingqing Liu is determining, first, what metabolic or signaling pathway(s) downstream of adiponectin receptors leads to this rise in 5’AMP. Second, she is determining whether this rise in 5’AMP is necessary for activation of AMPK. Activation of long-chain fatty acids to the CoA derivative is one important metabolic process that directly generates 5’AMP, and at least some Acyl CoA synthase isoforms are on the plasma membrane. She therefore proposes that one or more Acyl CoA synthase enzymes are directly coupled to adiponectin receptors, possibly to T- cadherin. Her recent data show that free fatty acids, essential substrates for the production of 5’AMP by acyl-CoA synthases, are required for AMPK activation by adiponectin in C2C12 myocytes. She further demonstrated that Acsl1 and FATP1 are the two major isoforms expressed in skeletal muscle. Free fatty acids are also required for AMPK activation by adiponectin and adiponectin stimulates long-chain fatty acid uptake in 3T3 L1 adipocytes. FATP1 and Acsl1, the two major acyl-CoA synthase isoforms in adipocytes, are, as she showed, essential for activation of AMPK by adiponectin.
Previous work showed that after 30-60 min insulin induces plasma membrane translocation of FATP1 in 3T3 L1 adipocytes. Recently Qingqing showed that after 40-60 min insulin also increases the AMP/ATP ratio and activates the AMPK signaling pathway in 3T3 L1 adipocytes. FATP1 and Acsl1 are also important in these processes. The activation of AMPK by insulin occurs only at 40 min, versus that of adiponectin, which peaks at 5 min, suggesting that the two hormones use distinct signaling pathways to activate acyl-CoA synthases.
She is currently testing the hypothesis that adiponectin enhances Acyl CoA synthase activity by stimulating its translocation from intracellular compartments to the plasma membrane. She is also determining the detailed molecular mechanism involved in insulin stimulation of AMPK and comparing it to that used by adiponectin.
Adiponectin receptors The signaling receptors for adiponectin are not known. Several years ago Christopher Hug, assisted by Jin Wang, used an expression cloning strategy to identify T- cadherin as a receptor for hexameric and high molecular weight forms of adiponectin. T-cadherin is highly and specifically expressed in the vasculature, where it is predominantly found in endothelial and smooth muscle cells in the blood vessel intima. At its C-terminus T-cadherin is attached to the plasma membrane via a GPI anchor. Chris’ studies indicate that T-cadherin is the major adiponectin binding protein in the body, as deletion of T-cadherin results in a many-fold increase in the level of all isoforms of adiponectin in the circulation. Immunohistochemical localization of adiponectin demonstrated that mice lacking T-cadherin had no detectable binding of adiponectin to the vascular endothelium, in contrast to wild-type animals that had substantial binding of adiponectin to the endothelium. T- cadherin is upregulated following vascular injury and Chris hypothesizes that, by binding to adiponectin, it may play a role in atherosclerosis progression as well as blood vessel formation and endothelial cell function.
Furthermore, T-cadherin null mice demonstrate hepatic insulin resistance, a phenotype virtually identical to that of adiponectin knockout animals. In conjunction with Dr. Gerry Shulman’s lab at Yale Medical School, Chris has characterized the metabolic and physiologic abnormalities of mice lacking T-cadherin, which may mimic those of the metabolic syndrome. Using the hyperinsulinemic euglycemic clamp technique on mice fed a high-fat diet for three weeks, they demonstrated that after an overnight fast there was no difference in insulin stimulated whole body glucose uptake, glycolysis, and glycogen synthesis. However, hepatic glucose production rates in T-cadherin deficient animals during the clamp were significantly higher than those of the control animals. Thus, T-cadherin deficient mice demonstrate hepatic, but not peripheral, insulin resistance. These results confirm that T-cadherin is a bona fide receptor for high-molecular weight forms of adiponectin, and that loss of T-cadherin causes a metabolic phenotype similar to that reported for loss of adiponectin. Currently, in his own laboratory at Children’s Hospital Boston and Harvard Medical School, Chris is determining the role of T-cadherin in adiponectin activation of the AMPK and NF-kB signal transduction pathways, and is studying the downstream pathways activated by adiponectin binding to T-cadherin.
Adiponectin and development of mammary and other cancers Both in mice and humans the serum levels of adiponectin are inversely correlated with adipose mass and body mass index (BMI). Epidemiological studies have demonstrated that serum adiponectin levels have an inverse association with breast cancer risk; low serum adiponectin concentrations, such as occur during obesity, are associated with large tumors and tumors of high histological grade. Moreover, adiponectin inhibits growth of several cell types, including vascular smooth muscle cells and several breast cancer lines, although the signal transduction pathways it activates and the mechanism(s) by which it exerts these anti-proliferative effects are unknown. Therefore, it is important to reveal the role of adiponectin in breast cancer formation and progression.
Yutong Sun hypothesizes that low serum level of adiponectin will accelerate mammary tumor formation and lead to larger tumors in mice, and that adiponectin can inhibit breast tumor cell proliferation, survival and migration/invasion. In order to determine whether adiponectin deficiency can decrease the latency and/or increase the incidence of breast cancer formation in mice, Yutong has bred breast cancer mouse models (MMTV-Her2/neu transgenic mice, and MMTV-Cre/Flox neo NeuNT mice) into an adiponectin -/- background. He has also introduced DMBA, a carcinogen into adiponectin -/- and wild- type. Currently, he is characterizing mammary tumor formation in these mice.
Furthermore, employing tumor cell implantation, Yutong is examining whether adiponectin deficiency affects tumor growth, angiogenesis, and metastasis in mice. Mouse melanoma cells (B16F10) and Lewis Lung Carcinoma cells (LLC1) were injected subcutaneously into the back of adiponectin knockout and control C57BL/6J mice. Both tumors grew faster in adiponectin knockout mice; by 14 days, the average tumor volume in adiponectin knockout mice was 2-3 fold that in control mice. To determine the role of adiponectin in metastasis, B16F10 cells were injected into adiponectin knockout and control C57BL/6J mice through the tail vein, and nodules in lungs were counted 10 days later. Results indicate that adiponectin does not affect B16F10 metastasis.
Immunohistochemistry staining of the tumor sections indicates that adiponectin deficiency does not affect tumor cell mitosis or apoptosis. Although several reports showed that adiponectin might play either an inhibitory or a stimulatory role in angiogenesis, IHC staining on tumor sections with the antibody MECA-32, a marker for endothelial cells, suggests that adiponectin has no effects on tumor- induced angiogenesis. Tumors from adiponectin- deficient mice do have less macrophage infiltration than those from control mice. Using recombinant adiponectin protein, which was purified from the conditioned medium of HEK293 cells stably expressing adiponectin, Yutong demonstrated that adiponectin does not affect B16F10 cell proliferation in vitro. Currently, he is exploring the mechanisms by which tumors grow faster in adiponectin knockout mice and he will continue to examine whether adiponectin deficiency affects breast tumor growth, angiogenesis and metastasis in mice.
Adiponectin paralogs Guang William Wong, with the assistance of Claire Kitidis, used multiple genomic approaches to identify a family of ten highly conserved human and mouse adiponectin paralogs. These are designated as C1q/TNF-alpha related proteins (CTRP)-1 to 10.
Of all the CTRP paralogs, the highly- conserved CTRP9 shows the highest degree of amino acid identity to adiponectin in its globular C1q domain. CTRP9 is expressed predominantly in adipose tissue and female expresses higher levels of the transcript compared to male mice. CTRP9 is a secreted glycoprotein with multiple posttranslational modifications in its collagen domain that include hydroxylated prolines and hydroxylated and glycosylated lysines. It forms and is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum. Furthermore, CTRP9 and adiponectin are secreted as hetero-oligomers when co-transfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally co-immunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, using gel filtration chromatography combined with co-immunoprecipitation analysis, they showed that adiponectin and CTRP9 form heterotrimers. Because different oligomeric forms of adiponectin have distinct biological activities, identification of CTRP9 that can heterotrimerizes with adiponectin impacts the study of adiponectin function. In cultured differentiated myotubes CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathway. And adenovirus-mediated overexpression of CTRP9 significantly lowered serum glucose levels in obese (ob/ob) mice compared to controls. Collectively, these results suggest that CTRP9 is a novel adipokine and further study of CTRP9 will yield novel mechanistic insights into its physiologic and metabolic function.
An understanding of the natural metabolic functions of CTRP9 and the other orthologs will likely emerge from analysis of the CTRP- overexpressing transgenic mice and CTRP gene knock- out mice Guang is now generating. Guang is also using expression cloning strategies to identify the receptors for these novel proteins. Much of this work is continuing in Guang’s laboratory at the Johns Hopkins School of Medicine.
Insulin resistance Alice Lo is a graduate student in Ernest Fraenkel's lab in the Biological Engineering Department at MIT. She is interested in adopting a systems approach in understanding TNF-alpha induced-insulin resistance in adipocytes and how certain pharmacological agents are able to ameliorate the condition. Using the 3T3-L1 adipogenesis model we have used extensively, she is studying how particular transcription factors and coregulators change their expression and genome-wide binding under different conditions. She is going to correlate these changes with changes in expression of their targets and with functional measurements including insulin sensitivity and adipokine secretion. A knockdown approach will be used to investigate the role of some transcription factors and coregulators in mediating insulin sensitivity/resistance.
| E. Regulated cleavage and release of the extracellular domain of transmembrane precursors of several secreted growth factors |
Protease cleavage and release of the extracellular domain (ECD, "ectodomain shedding") of a multitude of transmembrane proteins has been linked to the activation of many signaling pathways including the MAPK pathway. Cleavage of the ECD is mostly carried out by metalloproteases (MMPs) of the ADAM family (“a disintegrin and metalloprotease”). ECD cleavage is often followed by and is a prerequisite for intramembranous cleavage of the intracellular domain (ICD) of the same protein by gamma-secretase; some of the cleaved ICDs translocate to the nucleus, where they may regulate gene transcription. Membrane-spanning pro-hormone ligands of the epidermal growth factor receptor (HER) family are well-studied examples of proteins that undergo ectodomain shedding and are physiologically important in many cellular contexts in organisms from Drosophila to mammals. But how the ectodomain cleavage machinery is regulated is largely unknown, as only a few specific stimuli that induce ectodomain shedding have been identified. As example, prolonged activation of the cardiac beta-adrenergic receptor leads to HB-EGF-cleavage, release of soluble HB-EGF, and development of cardiac hypertrophy. Andreas Herrlich showed that another HER-ligand, neuregulin1beta (NRG1-beta), is cleaved by an MMP in response to hypertonic stress and subsequently activates EGF- family receptors in an autocrine fashion. This signaling step leads to MAPK activation followed by enhanced expression of genes encoding water channels (aquaporins). Regulation of ectodomain cleavage could occur at least two levels - at the level of the MMP or via covalent modifications of the target protein, such as phosphorylation or ubiquitination on the cytosolic face.
Andreas, with the assistance of Karen Dubbin and Michelle Dang, two MIT undergraduates, is cloning novel genes that regulate ectodomain shedding using a high-throughput lentiviral shRNA gene knock-down strategy. They can detect cleavage of all chosen HER-ligands either by hypertonic stress, phorbol ester addition, or stimulation with lysophosphatidic acid in a FACS-based assay using mouse or human cell lines stably expressing one of the chosen pro-hormone ligands. The ligands are tagged at the extracellular domain with one of several epitope tags; at their cytosol-facing C- termini the proteins have been fused with EGFP. The extracellular epitope of the transmembrane pro-hormone ligand is detected with a fluorochrome-coupled (red) anti-epitope antibody, while the intracellular domain of the EGFP- fusion is detected by green fluorescence. Stimulation of cleavage results in a decrease of the red to green fluorescence ratio, while inhibition of basal or induced cleavage is reflected by an increase in this ratio. Andreas’ initial studies with this system showed that, when expressed in mouse lung epithelial cells, ectodomain cleavage of these three EGF ligands is specifically triggered by different stimuli and involves different PKC isoenzymes.
Studies utilizing inhibitors of protein kinase C isoenzymes or metalloproteinase inhibition by batimastat showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. Andreas, Karen and Michelle are now using this assay system for a 96 well plate high-throughput shRNA gene knockdown screen. Currently they are in the late stages of testing the effect of shRNA constructs targeting about 95% of all known mammalian kinases and phosphatases on TPA-induced ectodomain cleavage of TGF-alpha. By stimulating cleavage to only about 50% of total possible cleavage they can detect both decreases (inhibitors of cleavage) and increases (activators of cleavage) in the red: green fluorescent ratio in the same screen. Once candidate genes are identified they will also be tested in the context of the other physiological cleavage stimuli, hypertonic stress and GPCR stimulation. Further experiments to test the physiological significance of the candidate genes will be carried out in a breast cancer and kidney disease cell culture model system, both of which have relevant connections to EGF ligand cleavage.
Additionally, Andreas and Karen are determining which ADAM metalloprotease is responsible for the osmotic stress-dependent cleavage of EGF ligands by using mouse-embryonic fibroblasts that are either wild type or knock-out for certain ADAMs.